Method for isolating nucleic acids

ABSTRACT

It was found that adding a chelating agent during resuspension considerably increases the nucleic acid yield as the formation of precipitates which irreversibly adhere to the container wall is considerably reduced.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 15/596,275 filed May 16, 2017, which is acontinuation application of U.S. application Ser. No. 14/238,625 filedFeb. 12, 2014, now issued as U.S. Pat. No. 9,695,465, which is a U.S.national phase application of PCT/EP2012/065819 filed Aug. 13, 2012,which claims priority to EP Application No. 11177426.1 filed Aug. 12,2011. U.S. application Ser. Nos. 15/596,275 and 14/238,625 are hereinincorporated by reference in its entity.

The work leading to this invention has received funding from theEuropean Community's Seventh Framework Programme (FP7/2007-2013) undergrant agreement n° 222916.

FIELD OF THE INVENTION

The present invention pertains to the field of isolating nucleic acids,in particular the isolation of RNA, from biological samples, inparticular from a blood sample, with high yield and integrity.

BACKGROUND OF THE INVENTION

Several methods for isolating nucleic acids such as RNA and/or DNA areknown in the prior art that are based on different principles. Examplesof common nucleic acid isolation methods include but are not limited toextraction, solid-phase extraction, phenol-chloroform extraction,chromatography, precipitation and combinations thereof. Very common arenucleic acid isolation methods which involve the use of chaotropicagents and/or alcohol in order to bind the nucleic acids to a solidphase, e.g. a solid phase comprising or consisting of silica. Theisolation of RNA is particularly challenging, because RNAses areomnipresent in rather high amounts and are active over a broadtemperature range and usually do not need co-factors for their activity.Therefore, it is a challenge to provide a RNA isolation method whichprovides the RNA with good yield and quality.

Furthermore, in many fields such as e.g. the diagnostic field it isdesirous or even mandatory to isolate the nucleic acids from a largenumber of samples. For this purpose it is common to use automatedprocesses (wherein e.g. many samples are processed at the same time). Toassist the user and to reduce hands-on-time, robotic systems arecommonly used that can process a large number of samples in parallel.Usually, the samples are prepared manually for nucleic acid isolationand are then entered into the robotic system. Respective manualpreparation steps are e.g. common for stabilised blood samples.Respective manual steps include e.g. the centrifugation of thestabilised sample to generate a nucleic acid containing pellet and theresuspension of the pellet e.g. in a resuspension buffer (therebyreducing the sample volume). The respectively resuspended samples arethen ready e.g. for sample digestion and isolation and are placed intothe robotic system. Examples of commercially available robotic systemsthat operate according to this or a similar principle include but arenot limited to QIAsymphony (QIAGEN), QIAcube (QIAGEN) and MagnaPure 96(ROCHE).

Even though these robotic systems provide remarkable advantages whenprocessing a large number of samples, they also have certainlimitations. E.g. said robotic systems can usually only process acertain number of samples at one time. Said number is often lower thanthe number of samples that is manually prepared as one batch for nucleicacid isolation. Thus, not all of the prepared samples can be processedat the same time. This has the effect that the samples prepared fornucleic acid isolation often have different holding times between theirpreparation for nucleic acid isolation (e.g. the centrifugation andresuspension of the pellet as described above) and the actual nucleicacid isolation. While the first batch of the prepared samples isprocessed in the robotic system, the other prepared samples are put onhold. It was found that variations in the holding time of the preparedsample can influence the quality of the isolated nucleic acid as well asthe nucleic acid yield. During longer holding times, a portion of thenucleic acids can form precipitates which irreversible stick to thecontainer and thus, can not be purified. Furthermore, the integrity ofthe nucleic acids, in particular of RNA, can be corrupted. Thus, thenucleic acid quantity and/or quality of the second and subsequent batchof the prepared samples that are processed in the robotic systems areoften lower. Hence, a longer holding time may reduce the quality and/orthe quantity of the isolated nucleic acids. This in particular poses aproblem if complex samples are processed, such as e.g. blood or samplesderived from blood and/or samples that were stabilised using a specificchemistry. This problem is further aggravated if large sample volumes(e.g. 1.5 ml and more) are processed. This loss in yield and/orintegrity can pose problems in particular in sensitive applicationfields such as e.g. the diagnostic field wherein an uniform nucleic acidisolation with respect to yield and quantity is important and hence,variations due to the used nucleic acid isolation method must beavoided.

Furthermore, methods that use magnetic beads as solid phase for bindingand isolating the nucleic acids often show a reduced nucleic acid yieldcompared to comparable methods that use a nucleic acid binding membraneinstead. Therefore, a loss in nucleic acid yield due to the precipitateformation has an even stronger impact on respective methods and systemthat use magnetic particles as nucleic acid binding solid phase.

Hence, there is a need in the state of the art to provide a nucleic acidisolation protocol which provides comparable high nucleic acid yieldsand also a high yield of small nucleic acids, even if the holding timesvary between samples and also during extended holding times, a highnucleic acid, in particular RNA integrity.

Therefore, it is an object of the present invention to provide animproved method for isolating a nucleic acid, in particular RNA, from asample, in particular a blood sample. Furthermore, it is the object ofthe present invention to provide a method that allows the isolation ofnucleic acids from a plurality of samples with a comparable qualityand/or quantity, even if the holding times between the preparation ofthe samples for isolation and the actual isolation of the nucleic acidsvaries.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that variationsin the nucleic acid yield and quality can be significantly reduced if atleast one chaotropic agent and at least one chelating agent are includedin the sample that is prepared for nucleic acid isolation. It was foundthat including at least one chaotropic agent and at least one chelatingagent into the sample prepared for nucleic acid isolation has theadvantage that the quality and yield of the nucleic acids that areisolated from respectively prepared samples is substantially maintainedeven if the prepared samples stand for a prolonged period of time (e.g.selected from 0.5 h to 12 h, 0.75 h to 11 h, 1 h to 10 h, 1.25 h to 9 h,1.5 h to 8.5 h, 1.75 h to 8 h, 1.75 h to 7.5 h, 1.25 h to 7 hours, 1.5 hto 6.5 h, 1.75 h to 6 h, 2 h to 5.5 h) before the nucleic acids areactually isolated from the respectively prepared samples. This advantageis particularly important when processing a large number of samplesbecause in this case variations in the holding time between samples arerather common, in particular when using an automated system. However,this advantage is also generally important as it reduces the need todirectly isolate the nucleic acids from the samples that are preparedfor nucleic acid isolation. This provides more flexibility.

According to a first aspect, the present invention provides a method forisolating nucleic acids, preferably RNA, from a sample, preferably ablood sample, comprising the following steps:

-   -   a) obtaining a sample which has been stabilised by the use of at        least one cationic detergent, wherein the cationic detergent has        formed complexes with the nucleic acids;    -   b) obtaining the complexes optionally together with other sample        components from the stabilised sample, wherein said complexes        comprise the nucleic acids to be isolated;    -   c) resuspending the complexes and optionally adding one or more        additives before, during and/or after resuspension, thereby        obtaining a resuspended sample comprising at least    -   i) the nucleic acids to be isolated;    -   ii) at least one chaotropic agent; and    -   iii) at least one chelating agent; and    -   d) isolating nucleic acids from the resuspended sample.

As discussed above, the method according to the present invention allowsto isolate nucleic acids with comparable good yield and quality fromsamples, even if the holding time between step c) and step d) shouldvary. The addition of the chaotropic agent preserves the quality of thenucleic acids contained in the resuspended sample, in particular of RNA.However, adding a chaotropic agent has the drawback that the nucleicacids precipitate during longer holding times and furthermore, theprecipitated nucleic acids may irreversibly stick to the walls of thecontainer which comprises the sample what considerably reduces thenucleic acid yield. The inventors now surprisingly found that theincorporation of a chelating agent in the resuspended sampleconsiderably reduces the formation of precipitates and furthermore,prevents the adherence of the precipitate to the walls of the containercomprising the resuspended sample which comprises the chaotropic agent.Thereby, a loss in the nucleic acid yield can be efficiently reduced.Thus, the teachings of the present invention allow the efficientisolation of nucleic acids, in particular RNA, with comparable yield andquality even if the resuspended samples prepared for nucleic acidisolation as described above are not directly processed but havedifferent and/or prolonged holding times.

According to a second aspect, a method is provided for isolating nucleicacids from a sample, preferably a blood sample, wherein the nucleicacids are isolated from a plurality of samples and wherein variations inyield and quality of the nucleic acids that are isolated from saidplurality of samples which result from that the plurality of samplesprepared for isolation have diverging holding times before the nucleicacids are isolated from the prepared samples are thereby reduced thatthe samples prepared for isolation comprise at least one chaotropicagent and at least one chelating agent.

According to a third aspect, the present invention pertains to the useof a chelating agent in order to prevent or reduce the formation of aprecipitate that attaches to the container wall of a sample comprisingat least one chaotropic agent and nucleic acids. As discussed above,prolonged holding times of respective samples result in the formation ofa precipitate, that sticks to the container wall comprising thechaotropic agent containing sample, thereby severely reducing thenucleic acid yield. Thus, the addition of the chelating agent as istaught by the present invention has the advantageous effect that theformation of a respective precipitate is reduced or even completelyprevented. Thus, nucleic acids can be isolated with good yield andquality, even if the holding time between the preparation of the samplesfor isolation and the actual isolation is increased. This providesparticular advantages if the technology of the present invention is usedin order to isolate nucleic acids in automated processes wherein a largenumber of samples is prepared for nucleic acid isolation but whereinonly a portion of the prepared samples can be processed batchwise on theautomated system. Thus, the teachings of the present invention ensuresubstantially uniform isolation results with respect to quality andyield even if the holding time between different samples varies and/oris prolonged, what is in particular advantageous for challenging fieldssuch as the isolation of nucleic acids for the medical and/or diagnosticfield.

Other objects, features, advantages and aspects of the presentapplication will become apparent to those skilled in the art from thefollowing description and appended claims. It should be understood,however, that the following description, appended claims, and specificexamples, while indicating preferred embodiments of the application, aregiven by way of illustration only. Various changes and modificationswithin the spirit and scope of the disclosed invention will becomereadily apparent to those skilled in the art from reading the following.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing RIN values (RNA integrity number) of RNAisolated from blood samples as described in Example 1.

FIG. 2 is a graph showing yield of nucleic acids isolated from bloodsamples as described in Example 2.

FIG. 3 shows the effects of resuspension steps on precipitate formationat the bottom of test tubes as described in Example 3.

FIG. 4 is a graph showing yields of nucleic acids isolated from bloodsamples as described in Example 3.

FIG. 5 is a graph showing RIN values of RNA isolated as described inExample 3.

FIG. 6 is a graph showing C_(T) values obtained with the MISCRIPT®primer assay (hsa-miR30b) when analyzing eluates obtained as describedin Example 3.

FIG. 7 shows the mean yield of total RNA isolated according to Example4.

FIG. 8A shows bulk precipitates stuck at the bottom of test tubes forsamples resuspended without EDTA as described in Example 5.

FIG. 8B shows no precipitates at the bottom of test tubes for samplesresuspended with EDTA as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that differences innucleic acid quality and yield that are attributable to differentholding times between sample preparation and actual nucleic acidisolation can be reduced by incorporating at least one chaotropic agentand at least one chelating agent into the sample that is prepared fornucleic acid isolation.

According to a first aspect, the present invention provides a method forisolating nucleic acids from a sample, preferably a blood sample,comprising the following steps:

-   -   a) obtaining a sample which has been stabilised by the use of at        least one cationic detergent, wherein the cationic detergent has        formed complexes with the nucleic acid;    -   b) obtaining the complexes optionally together with other sample        components from the stabilised sample, wherein said complexes        comprise the nucleic acid to be isolated;    -   c) resuspending the complexes and optionally adding one or more        additives before, during and/or after resuspension, thereby        obtaining a resuspended sample comprising at least        -   i) the nucleic acid to be isolated;        -   ii) at least one chaotropic agent; and        -   iii) at least one chelating agent; and    -   d) isolating nucleic acid from the resuspended sample.

The individual steps of the method will be explained in further detail:

In step a) a sample is obtained which has been stabilised by the use ofat least one cationic detergent. E.g. the sample can be stabilised bycontacting the sample with at least one cationic detergent. The cationicdetergent supports the lysis the cells contained in the sample and formscomplexes with the released nucleic acid. The nucleic acids are therebystabilised. The use of cationic detergents for stabilising a sample, inparticular a blood sample, is described in the prior art (see e.g. EP 1031 626 and WO 02/00599, herein incorporated by reference) and will bedescribed in further detail below. Respective stabilised samples can bee.g. obtained at one facility, e.g. a hospital and shipped to a secondfacility, e.g. a laboratory, wherein the samples are further processedand wherein the nucleic acids are isolated from the stabilised sample.

In step b) the complexes are obtained from the stabilised sample. Saidcomplexes comprise the nucleic acid to be isolated. Said complexes alsocomprise the cationic detergent that was used for stabilising thesample. Said complexes can be obtained e.g. by sedimentation orfiltration. Thereby, usually other sample components such as proteinsand cell debris are also obtained together with the nucleic acidcontaining complexes. Preferably, the complexes are obtained in form ofa pellet. A respective pellet can be e.g. obtained by centrifuging thesample and discarding the supernatant thereby obtaining a samplecomprising the complexes comprising the nucleic acids and the cationicdetergents. Depending on the type of sample to be processed, therespective pellet usually also comprises further sample components suchas proteins and/or cell debris. This is in particular the case whenprocessing complex samples such as e.g. whole blood or samples derivedfrom blood such as serum or plasma. Suitable methods for separating thecomplexes e.g. by obtaining them in form of a pellet from the stabilizedsamples are also described in EP 1 031 626 paragraph [51] et seq, hereinincorporated by reference. Obtaining the complexes, e.g. in form of apellet, has the advantage that the subsequent nucleic acid isolationthat is performed in step d) can be performed in smaller volumina whatis cost-efficient as less reagents are necessary for nucleic acidisolation and furthermore, provides an important advantage when usingautomated methods for nucleic acid isolation because many automatedsystems are limited with respect to the volume they can process.

In step c) the complexes and optional other sample components, which arepreferably obtained in form of a pellet, are resuspended, therebyobtaining a resuspended sample. Said resuspended sample comprises thenucleic acid to be isolated, the cationic detergent and optionallyfurther sample components such as e.g. proteins and/or cell debris thatwere collected together with the complexes. Furthermore, optionallyfurther additives can be added either before, during or afterresuspension such as e.g. a chaotropic agent and/or a protein degradingcompound. Herein, we refer to the resuspended complexes includingoptional further sample components and/or optional additives that wereadded before, during and/or after resuspension as “resuspended sample”.According to the invention, the resuspended sample comprises at leastone chaotropic agent. Preferably, the chaotropic agent is added asseparate additive after the complexes were resuspended to provide aresuspended sample comprising a chaotropic agent. This order ispreferred because the chaotropic agent might hamper the actualresuspension process of the complexes, in particular if the complexesare obtained in form of a pellet. Preferably, the chaotropic agent isadded to the resuspended complexes in form of an aqueous solution as isdescribed below in order to generate a resuspended sample comprisinginter alia the nucleic acids and the chaotropic agent. As discussedabove, the chaotropic agent protects the nucleic acids, in particularRNA, from degradation, thereby increasing the quality of the isolatednucleic acids. As is shown in the examples, if a chaotropic agent iscomprised in the resuspended sample this has the effect that nucleicacids, in particular RNA, can be obtained with comparable quality evenif the holding time varies and e.g. is extended between step c) and stepd). Therefore, the isolation results obtained for different sampleshaving different holding times between step c) and step d) are moreuniform and thus comparable when using the method according to thepresent invention. However, the chaotropic agent not only has beneficialeffects with respect to the preservation of the nucleic acid integrity,but also causes problems. During longer holding times it causes theformation of precipitates that can stick to the wall (the term “wall”also includes the bottom) of the container which comprises theresuspended sample. This precipitate formation and adherence to thecontainer wall that is observed if a chaotropic agent is included in theresuspended sample reduces the yield of nucleic acids (DNA and/or RNA)that can be isolated from the resuspended sample. As is shown by theexamples, the yield can even be reduced by up to 30% or even more, ifthe resuspended samples rest for a prolonged holding time between stepc) and step d). This problem is overcome by the teachings of the presentinvention by additionally including at least one chelating agent in theresuspended sample. The chelating agent surprisingly reduces theformation of insoluble precipitates and also reduces the adherence ofsaid precipitates to the container wall, thereby efficiently reducingthe loss of nucleic acids during extended holding times between step c)and step d). The chelating agent can be added prior, during or afterresuspension of the complexes in order to reduce respectively preventthe formation of precipitates that reduce the nucleic acid yield.Preferably, the chelating agent is added prior to the addition of thechaotropic agent. If the chelating agent is added after resuspension ofthe complexes, it should be added basically directly after resuspensionin order to ensure that it can exhibit its beneficial effects describedabove. It should be added within one hour, preferably within 30 min,more preferably within 15 min, more preferably within 10 min, morepreferably within 5 min, most preferably within 3 min.

Optionally, the method may comprise further treatment steps in order toprepare the resuspended sample for step d). Examples of respectiveadditional steps are described in further detail below.

In step d), the nucleic acids are isolated from the resuspended sample.Here, basically any nucleic acid isolation method can be used. Suitablenucleic acid isolation methods are known in the prior art and includebut are not limited to extraction, solid-phase extraction, silica-basedpurification methods, nucleic acid isolation procedures using chaotropicagents and/or at least one alcohol and a nucleic acid binding solidphase, magnetic particle-based purification, phenol-chloroformextraction, chromatography, anion-exchange chromatography (usinganion-exchange surfaces), electrophoresis, filtration, precipitation,chromatin immunoprecipitation and combinations thereof. Preferably, thenucleic acids are isolated in step d) using an automated system.Preferably, the nucleic acids are isolated from a plurality of samples.The plurality of samples can be processed batchwise. The holding timemay vary between individual batches, because the present inventionensures comparable nucleic acid isolation results even if the holdingtime significantly varies between batches. Suitable methods and holdingtimes are also described below.

The advantages of the respective method were explained above and arealso demonstrated by the examples. A loss in quality and/or quantity ofthe isolated nucleic acids that are attributable to longer holding timesbetween step c) and step d) can be efficiently avoided due to themodified resuspension step taught by the present invention. Therefore,the present method provides more flexibility with respect to theallowable holding times between steps c) and d) what is in particularbeneficial if the method according to the present invention is performedon an automated system, wherein, preferably, a plurality of samples areprocessed in a batchwise procedure.

Preferred embodiments of the method according to the present inventionand of the steps a) to d) are described subsequently.

According to one embodiment, a resuspension solution is added in step c)to the obtained complexes wherein said resuspension solution comprises asalt, preferably a non-chaotropic salt. As salt, several salts can beused including but not being limited to ammonium salts and alkali metalsalts, preferably ammonium acetate, ammonium sulphate, KCl or NaCl.Preferably, an ammonium salt is used. Preferably, the chelating agent iscomprised in the resuspension solution. This embodiment is easy inhandling and furthermore, also ensures that the chelating agent canimmediately exhibit it's beneficial effects on the resuspended sample inthat it prevents the formation of precipitates and/or adherence of thenucleic acids to the container walls when the resuspended sample whenthe chaotropic agent is added in order to protect the nucleic acid. Asdiscussed above, it is preferred to add the chaotropic agent after thecomplexes were resuspended and accordingly, after the resuspensionsolution was added. Accordingly, the resuspension solution preferablydoes not comprise a chaotropic agent, in particular does not comprise achaotropic salt and accordingly, is a non-chaotropic resuspensionsolution. According to one embodiment, the resuspension solutioncomprises the chelating agent in a concentration of at least 1 mM, atleast 5 mM, preferably at least 7.5 mM, more preferred at least 10 mM,at least 15 mM or at least 20 mM. Preferably, the concentration range isselected from 1 mM to 150 mM, 5 mM to 100 mM, 5 mM to 75 mM, 7.5 mM to65 mM, 7.5 mM to 50 mM and 10 mM to 30 mM. As was shown by examples,already a low concentration of a chelating agent such as EDTA issufficient in order to achieve the beneficial effect on the precipitateformation. In some embodiments to use a lower to medium concentration ofthe chelating agent e.g. in a range of 1 mM to 50 mM, preferably 5 mM to30 mM or 7.5 mM to 20 mM. To use lower concentrations of the chelatingagent reduces the risk that the chelating agent is carried over into theeluate while still achieving the beneficial effect on the precipitateformation. The chelating agent may also be added separately from theresuspension solution, e.g. in liquid or solid form.

According to one embodiment, the chelating agent is added in aconcentration so that the resuspended sample comprises the chelatingagent in a concentration of at least 0.5 mM, at least 2.5 mM, preferablyat least 3.5 mM, more preferred at least 5 mM, at least 7.5 mM or atleast 10 mM. Preferably, the chelating agent is added in a concentrationso that the resuspended sample comprises the chelating agent in aconcentration selected from 0.5 mM to 100 mM, 2.5 mM to 75 mM, 2.5 mM to60 mM, 3.5 mM to 50 mM, 3.5 mM to 30 mM, 3.5 mM to 25 mM, 3.5 mM to 20mM, 5 mM to 15 mM and 5 mM to 10 mM.

The chelating agent that is used according to the present inventionprevents or reduces the formation of a precipitate and/or the adherenceof precipitate to the container when comprised in the resuspendedsample. According to one embodiment, the chelating agent is an organicligand, which is capable of forming two or more separate coordinatebonds to a metal cation and comprises at least one nitrogen atom asnucleophilic coordinating atom. According to one embodiment, thechelating agent is an organic ligand, which is capable of forming two ormore separate coordinate bonds to a metal cation and comprises at leastfour nucleophilic coordinating atoms. According to one embodiment, thechelating agent is an organic ligand, which is capable of forming two ormore separate coordinate bonds to a metal cation and comprises at leastfour carboxylic groups. According to one embodiment, the chelating agentis an organic ligand, which is capable of forming two or more separatecoordinate bonds to a metal cation, the stability constant of theresulting complex being at least 10 ⁴ M⁻¹ in case of calcium as metalcation. Chelating agents according to the present invention include, butare not limited to diethylenetriaminepentaacetic acid (DTPA),ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraaceticacid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA). According to apreferred embodiment, EDTA is used. As used herein, the term “EDTA”indicates inter alia the EDTA portion of an EDTA compound such as, forexample, K₂EDTA, K₃EDTA or Na₂EDTA.

As discussed above, the chelating agent has several beneficial effects.It reduces the precipitate formation and in particular the binding ofthe nucleic acid containing precipitate to the container comprising theresuspended sample which comprises a chaotropic agent, thereby reducingor even preventing a loss in nucleic acid yield, in particular if theresuspended sample stands for a prolonged times before the nucleic acidsare isolated from the resuspended sample (e.g. more than 0.1 h, morethan 0.2 h, more than 0.3 h, in particular more than 0.5 h, more than0.75 h, more than 1 h, more than 1.25 h, more than 1.5 h, more than 1.75h, more than 2 h, more than 2.5 h or more than 3 h hours or in a rangefrom 0.1 h to 12 h, 0.5 h to 11 h, 0.75 h to 10 h, 1 h to 9 h, 1.25 h to8.5 h, 1.5 h to 8 h, 1.75 h to 7.5 h, 1.25 h to 7 hours, 1.5 h to 6.5 h,1.75 h to 6 h or 2 h to 5.5 h). Thereby, the chelating agent reducesvariations in the nucleic acid isolation efficiency and/or quantityattributable to different and in particular prolonged holding timesbetween step c) and d).

According to one embodiment, the chelating agent that is used to preventor reduce the formation of a nucleic acid containing precipitate and/orthe adherence of precipitate to the container when comprised in theresuspended sample is no classical buffering agent such as citrate.However, a buffering agent may be and is also preferably comprised inthe resuspended sample in addition to the chelating agent that is usedto prevent or reduce the formation of the precipitate and/or theadherence of precipitate to the container. A respective buffering agentmay be e.g. comprised in the resuspension solution.

As discussed above, in order to preserve the integrity of the nucleicacids comprised in the sample, in particular RNA, and in particularduring longer holding times of the resuspended sample, a chaotropicagent is incorporated in and thus comprised in the resuspended sample.This is particularly advantageous when isolating RNA which is verysensitive to the omnipresent RNases. As is demonstrated by the examples,incorporating a chaotropic agent into the resuspended sampleconsiderably improves the quality of the isolated RNA, in particular ifthe sample stands for a longer time between steps c) and d) as is oftenthe case when isolating nucleic acids from a plurality of samples usingan automated system and in particular, if a batchwise procedure is usedto process the plurality of samples.

Any chaotropic agent can be used in step c) that causes disorder in aprotein or nucleic acid by, for example, but not limited to altering thesecondary, tertiary or quaternary structure of a protein or a nucleicacid while leaving the primary structure intact. Preferably, achaotropic salt is used. Preferred chaotropic agents include but are notlimited to chaotropic salts comprising e.g. thiocyanate, isothiocyanate,perchlorate, trichloroacetate, trifluoroacetate or iodide and/orcomprising guanidinium and are preferably selected from guanidiniumhydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate,sodium thiocyanate, sodium iodide, sodium perchlorate, sodiumtrichloroacetate, sodium trifluroacetate, urea and the like. Preferably,the chaotropic agent is GTC or GITC or an equally strong chaotropicagent. Respective strong chaotropic agents are very efficient inprotecting the nucleic acid, in particular RNA, from degradation.However, respective strong chaotropic agents strongly induce theprecipitate formation and adherence of the precipitate to the containerwall described above. According to one embodiment, the resuspendedsample comprises the chaotropic agent in a concentration selected fromthe group consisting of 0.1 up to the saturation limit, 0.2 to 6 M, 0.1M to 4 M, 0.5 M to 3 M, 0.75 M to 2.5 M, most preferred at least 1 M. Asis shown in the examples, using a respective concentration is useful toefficiently preserve the integrity of the incorporated nucleic acids.The at least one chaotropic agent may be added in step c) in form of aseparate solution. Said separate solution preferably comprises achaotropic salt, e.g. a guanidinium salt, a buffer and/or a chelatingagent. Preferably, said buffer comprises sodium citrate.

The pH value of the resuspended sample preferably lies in a range thatis selected from 5 to 10, 5.5 to 9.5, 6 to 9, 6.5 to 8.5 and preferably7 to 8. In a pH range of 5 to 8.5, pellets obtained from blood sampleswere particularly well resuspended. Therefore, according to oneembodiment, a resuspension solution is added in step c) to thecomplexes, preferably the pellet comprising the cationic detergent andthe nucleic acids that which has a pH value that achieves, with theadded amount, a pH value in the resuspended sample that lies in theabove defined pH range. According to one embodiment, the pH value of theresuspension solution lies in a pH range that is selected from 5 to10.5, 5.5 to 10, 5.7 to 10, 6 to 9.7, 6.3 to 9.5, 6 to 9 and 6 to 8.5.Preferably, the pH range lies with a range of 5.7 to 10, more preferred6 to 9. Here, the achieved nucleic acid yield was optimal.

According to one embodiment, a protein degrading compound is added instep c) and thus is comprised in the resuspended sample. It was found bythe inventors that the addition of a protein degrading compound supportsthe effects of the chelating agent. Thus, including a protein degradingcompound in the resuspended sample is beneficial in order to prevent theprecipitate formation and the adherence of nucleic acid containingprecipitates to the container walls. According to a preferredembodiment, the protein-degrading compound is a proteolytic enzyme. Aproteolytic enzyme refers to an enzyme that catalyzes the cleavage ofpeptide bounds, for example in proteins, polypeptides, oligopeptides andpeptides. Exemplary proteolytic enzymes include but are not limited toproteinases and proteases in particular subtilisins, subtilases,alkaline serine proteases and the like. Subtilases are a family ofserine proteases, i.e. enzymes with a serine residue in the active side.Subtilisins are bacterial serine protease that has broad substratespecificities. Subtilisins are relatively resistant to denaturation bychaotropic agents, such as urea and guanidine hydrochloride and anionicdetergents such as sodium dodecyl sulfate (SDS). Exemplary subtilisinsinclude but are not limited to proteinase K, proteinase R, proteinase T,subtilisin, subtilisin A, QIAGEN Protease and the like. Discussions ofsubtilases, subtilisins, proteinase K and other proteases may be found,among other places in Genov et al., Int. J. Peptide Protein Res. 45:391-400, 1995. Preferably, the proteolytic enzyme is proteinase K.Incorporating a protein-degrading compound such as in particular aproteolytic enzyme in the resuspended sample has the advantage that theresuspended sample is also pre-digested during the holding time beforethe nucleic acids are isolated in step d). In non-limiting aspects, theproteolytic enzyme is comprised in the resuspended sample in aconcentration between about 0.05 mg/ml to about 10 mg/ml. In otherembodiments the range can be between from about 0.1 mg/ml to about 5mg/ml, or between about 0.2 mg/ml to about 1.0 mg/ml. It was found bythe inventors that the protein-degrading compound such as in particulara proteolytic enzyme supports the prevention of precipitate formationand adherence of precipitate to the container walls. This effect wasseen, even if the sample was not incubated under conditions that areoptimal for the performance of the proteolytic enzyme (e.g. heating andagitation).

In order to efficiently prepare the resuspended sample for the nucleicacid isolation in step d) it is preferred to thoroughly digest theresuspended sample prior to isolating the nucleic acids. Here, differentoptions exist that may be used in conjunction with the presentinvention. Some non-limiting options are subsequently described.

According to one embodiment, which is preferred, a proteolytic enzyme isincluded in the resuspended sample. As discussed above, the proteolyticenzyme may be directly added in step c) either before, during or after,preferably directly after, resuspension of the complexes and thereby isincluded in the resuspended sample. According to one embodiment, therespectively prepared resuspended sample is then incubated underconditions that allow at least the partial digestion of the sample.Digestion may e.g. occur during the holding time of the resuspendedsample e.g. at room temperature. Thereby, the resuspended sampleis—depending on the holding time—at least partially digested prior tostep d). Digestion may be incomplete during the holding time betweenstep c) and d). In order to ensure an efficient nucleic acid isolationin step d), a thorough digestion of the resuspended sample is preferred.Thus, according to one embodiment, after the holding time of theresuspended sample and thus, preferably in the initial step of thenucleic acid isolation performed in step d), the resuspended sample isincubated in step d) under conditions that support the digestion of theresuspended sample, preferably under heating and agitation for at least3 min, preferably at least 5 min. If no proteolytic enzyme has beenadded in step c), a proteolytic enzyme would be added in step d) inorder to allow an efficient digestion of the resuspended sample. It isalso within the scope of the present invention to add an additionalamount or a further (e.g. a different) proteolytic enzyme orprotein-degrading compound in step d) even if a proteolytic enzyme wasalready included in the resuspended sample in step c).

According to a preferred embodiment, a proteolytic enzyme is comprisedin the resuspended sample that is obtained in step c). During theholding time of the resuspended sample, the proteolytic enzyme can atleast partially digest the resuspended sample even under non-optimalincubation conditions (e.g. at room temperature). In order to ensure ahigh efficiency of the digestion, the resuspended sample is preferablyincubated after the holding time in the initial step of the nucleic acidisolation that is performed in step d) under conditions that promote thedigestion of the sample, e.g. heating and agitation.

According to one embodiment, the conditions that allow the digestion ofthe sample and which preferably are used in step d) comprise one or moreof the following

a) heating,

b) agitation,

c) the presence of salts,

d) a pH value of between 6 to 9 and/or

e) an incubation period of at least 3 min, preferably at least 5 min,most preferred for at least 10 min.

Preferably, said incubation step is performed under heating. Preferably,the resuspended sample is heated at least to a temperature of 35° C., atleast 40° C., or at least 50° C. and preferably is heated to atemperature of at least 55° C. during incubation. Using respectivehigher temperatures during incubation is in particularly favourable if aproteolytic enzyme such as proteinase K is used as protein-degradingcompound that shows its optimal, respective highest activity at highertemperatures. Under such conditions, the digestion of the resuspendedsample is promoted. Of course, a temperature should be used wherein theproteolytic enzyme is active. Furthermore, it is preferred that the saidincubation step is performed while agitating the resuspended sample.Non-limiting examples of agitation include shaking, stirring, mixing, orvibrating. In certain aspects, agitation comprises shaking. The shakingcan be one, two, or three dimensional shaking. A variety of shaking oragitating devices can be used. Non-limiting examples include theThermomixer (Eppendorf), TurboMix (Scientific Industries), Mo Bio VortexAdapter (Mo Bio Laboratories), Microtube holder vortex adapter(Troemner), and the Microtube foam rack vortex attachment (ScientificIndustries). Agitating can be performed for example in a mixer with atleast 50 rpm, at least 100 rpm, at least 200 rpm or at least 500 rpm.Preferably, heating and agitation is simultaneously performed, forexample by using a thermomixer or an equivalent apparatus that allowssimultaneous heating and agitation. When using at least one proteolyticenzyme as protein-degrading compound, incubation conditions are usedthat ensure that said enzyme works efficiently and is catalyticallyactive. The conditions depend on the proteolytic enzyme used and areknown, respectively determinable by the skilled person. Preferably, theincubation is performed in the presence of salts and/or ions thatpromote and/or maintain the activity of the proteolytic enzyme. Suitablesalts include but are not limited to NaCl, KCl, MgCl₂, or CaCl₂ orchaotropic agents such as chaotropic salts.

The above described conditions are particularly favourable when using aproteolytic enzyme as protein-degrading compound and said conditionspromote the digestion of the resuspended sample. As discussed above, adigestion of the resuspended sample under respective conditions ispreferably performed as initial step in nucleic acid step d).

Furthermore, as at least one chaotropic agent is included in theresuspended sample in order to preserve the integrity of the comprisednucleic acid, in particular the RNA, the digestion is performed in thepresence of at least one chaotropic agent, preferably a chaotropic salt.It is also within the scope of the present invention to add a furtheramount or type of chaotropic agent at the time wherein the digestion isperformed. For this purpose a digestion solution can be added whichcomprises at least one chaotropic agent, which may be the same ordifferent than the one(s) that is/are included in the resuspendedsample. Said digestion solution may also comprise additional compoundssuch as e.g. detergents and salts that promote the digestion and/orpreserve the comprised nucleic acid. Any chaotropic agent can be usedfor that purpose that causes disorder in a protein or nucleic acid by,for example, but not limited to altering the secondary, tertiary orquaternary structure of a protein or a nucleic acid while leaving theprimary structure intact. Preferred chaotropic agents that can be usedduring incubation with the at least one protein-degrading compound arechaotropic salts which include but are not limited to guanidiniumhydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate,sodium thiocyanate, sodium iodide, sodium perchlorate, sodiumtrichloroacetate, sodium trifluroacetate, urea and the like.

The incubation with the at least one protein-degrading compound todigest the resuspended sample is usually performed at a pH value thatdoes not lead to a degradation of the comprised nucleic acid.Furthermore, when using a proteolytic enzyme as protein-degradingcompound, a pH value should be used wherein the proteolytic enzyme isactive. Preferably, the incubation with the at least oneprotein-degrading compound for digesting the resuspended sample isperformed at a pH between 4.3 to 9, 6 to 8 and, preferably, is performedat a neutral pH value.

As discussed above, according to one embodiment, the proteolytic enzymeis included in the resuspended sample. In this embodiment, digestionalready occurs during the holding time of the resuspended sample betweenstep c) and d). However, as the resuspended samples may have differentholding times prior to step d) and furthermore, digestion might not becomplete under the conditions that are present during the holding time,it is preferred to additionally incubate the resuspended sample afterthe holding time under conditions that promote the digestion of thesample in the initial step of the nucleic acid isolation that isperformed in step d).

According to a preferred embodiment, the incubation for digesting theresuspended sample is performed under heating, agitation, in thepresence of chaotropic agents, a pH of 5 to 9, preferably 6 to 8,preferably a neutral pH, and for an incubation period of at least 3,preferably at least 5 minutes. In order to ensure efficient degradationof the proteins in step d), the resuspended sample should be incubatedin step d) for a period of at least 3 minutes, preferably at least 5minutes at elevated temperatures preferably above 50° C. in order toensure efficient protein degradation. According to a preferredembodiment, the incubation is performed for at least 5 minutes,preferably for at least 10 minutes.

After optionally, but preferably digesting the resuspended sample asdescribed above as initial step of the nucleic acid isolation procedurein step d), the nucleic acid can be e.g. bound to a solid phase and thenucleic acid can be optionally eluted therefrom. Preferred embodimentsare described below.

As solid phase, any material that is capable of binding nucleic acidsthat are present in or are released from a sample can be used andinclude a variety of materials that are capable of binding nucleic acidsunder suitable conditions. Exemplary solid phases that can be used inconjunction with the present invention include, but are not limited to,compounds comprising silica, including but not limited to, silicaparticles, silicon dioxide, diatomaceous earth, glass, alkylsilica,aluminum silicate, and borosilicate; nitrocellulose; diazotized paper;hydroxyapatite (also referred to as hydroxyl apatite); nylon; metaloxides; zirconia; alumina; polymeric supports, diethylaminoethyl- andtriethylaminoethyl-derivatized supports, hydrophobic chromatographyresins (such as phenyl- or octyl Sepharose) and the like. The term solidphase is not intended to imply any limitation regarding its form ordesign. Thus, the term solid phase encompasses appropriate materialsthat are porous or non-porous; permeable or impermeable; including butnot limited to membranes, filters, sheets, particles, magneticparticles, beads, gels, powders, fibers, and the like. According to oneembodiment, the surface of the solid phase such as e.g. the silica solidphase is not modified and is, e.g., not modified with functional groups.

According to a preferred embodiment, a solid phase comprising silica isused. Silica based nucleic acid isolation methods are broadly used inthe prior art. The solid phase comprising silica may e.g. have the formof a filter, fibres, membrane or particles. In particular preferred isthe use of silica particles that can be used in form of beads and whichpreferably have a particle size of about 0.02 to 30 μm, more preferred0.05 to 15 μm and most preferred of 0.1 to 10 μm. To ease the processingof the nucleic acid binding solid phase, preferably magnetic silicaparticles are used. The magnetic silica particles may e.g. beferrimagnetic, ferromagnetic, paramagnetic or superparamagnetic.Suitable magnetic silica particles are for example described in WO01/71732, WO 2004/003231 and WO 2003/004150. Other magnetic silicaparticles are also known from the prior art and are e.g. described in WO98/31840, WO 98/31461, EP 1 260 595, WO 96/41811 and EP 0 343 934 andalso include for example magnetic silica glass particles.

According to one embodiment, binding is performed under conditionshaving one or more, preferably at least two of the followingcharacteristics:

-   -   a) binding is performed in the presence of at least one        chaotropic agent,    -   b) binding is performed in the presence of at least one alcohol,    -   c) binding is performed in the presence of at least one        detergent,    -   d) binding is performed under conditions that promote binding of        the nucleic acids, in particular the RNA, and/or    -   e) binding is performed under conditions that promote binding of        small nucleic acids, in particular small RNA species.

According to one embodiment, the binding of the nucleic acids to thesolid phase is performed in step d) in the presence of at least onechaotropic agent, preferably a chaotropic salt and/or in the presence ofat least one alcohol. As discussed above, also a mixture of chaotropicagents can be used. The concentration of the chaotropic agent or mixtureof chaotropic agents that are used during binding may lie in a range of0.05 M up to the saturation limit. Preferred concentration ranges lie,depending on the chaotropic agent used, within 0.1 M to 7 M, 1 M to 7 M,1.5 M to 6 M and 2 M to 4 M. Suitable chaotropic agents are inparticular chaotropic salts and include but are not limited toguanidinium hydrochloride, guanidinium thiocyanate, guanidiniumisothiocyanate, sodium thiocyanate, sodium iodide, sodium perchlorate,sodium trichloroacetate, sodium trifluoroacetate, urea and the like andin particular preferred are guanidinium hydrochloride, guanidiniumthiocyanate and guanidinium isothiocyanate. As discussed above, theresuspended sample already comprises at least one chaotropic agent thathas been added in order to preserve the integrity of the nucleic acidsin the resuspended sample during longer holding times between step c)and step d). Thus, it is not necessary to additionally add a chaotropicagent in order to allow binding of the nucleic acids to the solid phase.However, it is also within the scope of the present invention toadditionally add at least one chaotropic agent in step d) e.g. in formof an aqueous binding solution.

As alcohol that can be used to promote binding, it is preferred to useshort chained branched or unbranched alcohols with preferably one to 5carbon atoms. Examples are methanol, ethanol, propanol, isopropanol andbutanol. Also mixtures of alcohol can be used. The alcohol is preferablyselected from isopropanol and ethanol, particularly well suitable isisopropanol when isolating RNA as target nucleic acid. Preferably, themethod according to the present invention does not involve the use ofphenol and/or chloroform.

The alcohol may be comprised in the binding mixture in a concentrationof 10% v/v to 90% v/v, in particular 15% v/v to 80% v/v, 20% to 80% v/v.The binding mixture in particular comprises the resuspended sample andthe solid phase and may optionally comprise further agents that wereadded to establish, respectively improve the binding conditions. Forisolating total RNA which also comprises small RNA, it is beneficial touse an alcohol concentration of ≥30% v/v, preferably ≥40% v/v to ≤90%v/v, more preferred ≥50% v/v to ≤90% v/v or ≥60% v/v to ≤80% v/v duringbinding and thus in the binding mixture. Respective higherconcentrations of alcohol improve the binding and thus the isolation ofshort nucleic acids (usually having a size respectively length of 500 ntor less), in particular small RNA species. Most preferred is an alcoholconcentration of ≥40% v/v to ≤90% v/v or ≥60% v/v to ≤80% v/v duringbinding when intending to isolate RNA which includes small RNA. Theseconcentrations work particularly well if the chaotropic agent(s) is/arepresent in higher concentrations and when binding the nucleic acids to asilica surface.

Thus, according to one embodiment, the isolation in step d) is performedusing binding conditions having one or more of the followingcharacteristics to bind the nucleic acids to a solid phase:

-   -   a) an alcohol concentration is used that is selected from the        group consisting of 10% v/v to 90% v/v, 15%v/v to 90% v/v, 20%        v/v to 85%v/v, 30%v/v to 80%v/v, 40% v/v to 85% v/v, 40%v/v to        80%, 40% v/v to 70%, ≥50% v/v to ≤80% v/v and ≥60% v/v to ≤80%        v/v,    -   b) a concentration of one or more chaotropic agents is used that        is selected from the group consisting of 0.05 M up to the        saturation limit, 0.1 M to 6 M and 1 M to 4 M, and/or    -   c) an alcohol concentration of at least 30% v/v, preferably at        least 40% v/v and at least one chaotropic agent is used for        binding RNA, including small RNAs to the solid phase.

To establish respective binding conditions, a binding solution whichcomprises e.g. the alcohol and the chaotropic agent can be added e.g. tothe resuspended sample, preferably to the resuspended digested sample.

Optionally, one or more detergents can be added to the binding mixtureto promote binding of the nucleic acid to the solid phase. Preferably,at least one ionic and/or at least one non-ionic detergent is added.Preferably, a non-ionic detergent is used in a concentration of at least5%. Said detergent can be added, e.g., together with the bindingsolution or can be provided by the resuspended sample and/or thedigestion solution if a respective digestion solution is added topromote the digestion, respectively lysis of the sample.

Furthermore, a buffer such as a biological buffer can be used forbinding, respectively can be incorporated in the binding solution.Non-limited examples of biological buffers include but are not limitedto HEPES, MES, MOPS, TRIS, BIS-TRIS Propane and others. Preferably, aTris buffer is used in the binding solution.

Therefore, according to one embodiment, the nucleic acid isolation instep d) comprises the addition of a binding solution which comprises atleast one alcohol and/or at least one chaotropic agent and optionally abiological buffer, preferably Tris, in order to establish the bindingconditions that allow to bind the nucleic acid that are comprised in theresuspended sample to the solid phase. Optionally, the binding solutionadditionally comprises a detergent as is described above. However, thecomponents can also be added separately to establish suitable bindingconditions in the binding mixture. Preferably, the binding solution pHis in a range that includes 8. According to one embodiment, the pH ofthe binding solution is in the range from pH 7.0 to 9, preferably 7.5 to8.5; most preferred the binding solution has a pH of 8.

According to one embodiment, one or more washing steps are performed inisolation step d) in order to further purify the isolated nucleic acids.According to one embodiment, one or more washing steps are performedwhile the nucleic acid is bound to the solid phase. For this purposecommon washing solutions may be used. According to one embodiment, thesolution used for washing comprises at least one chaotropic agent, atleast one alcohol, at least one detergent and/or at least one bufferingcomponent. Chaotropic agents that can be used in the washing solutionsinclude but are not limited to guanidinium hydrochloride, guanidiniumthiocyanate, guanidinium isothiocyanate and sodium iodide. Furthermore,chaotropic salts can be used which comprise a chaotropic anion selectedform the group consisting of trichloroacetate, perchlorate andtrifluoroacetate. Examples of respective chaotropic salts are alkalisalts like sodium perchlorate, sodium trichloroacetate and sodiumtrifluoroacetate. As alcohol, short chained branched or unbranchedalcohols with preferably one to 5 carbon atoms can be used for washing,respectively in the washing solution. Examples are methanol, ethanol,propanol, isopropanol and butanol. Preferably, isopropanol and/orethanol are used. Preferably, the washing solution comprises at least50% alcohol and at least 1 M chaotropic salt, preferably at least 2 Mchaotropic salt. Furthermore, the washing solution may comprise adetergent. Preferably, ionic and/or non-ionic detergents are used asdetergent. Preferably, a non-ionic detergent is used in a concentrationof at least 5%.

A further suitable washing solution which can be used alternatively oralso in addition to the washing solutions described above comprises analcohol and a biological buffer. Suitable alcohols and biologicalbuffers are described above. Preferably, isopropanol or ethanol, mostpreferred ethanol is used for this second washing step. Preferably,ethanol is used in a concentration of at least 70% v/v, preferably atleast 80% v/v. The biological buffer is preferably Tris at a pH ofapprox. 7 to 8. According to one embodiment, the solution used forwashing comprises at least one chaotropic agent, at least one alcohol,at least one detergent and/or at least one buffering component.

In case it is desired to perform an elution step to elute the nucleicacids from the solid phase, elution can be performed for example withclassical elution solutions such as water, elution buffers, inparticular biological buffers such as Tris and preferably elutionsolutions are used that do not interfere with the intended downstreamapplication. After elution, the eluate can be heat denatured. However,it is also within the scope of the present invention to release and thuselute the nucleic acids from the solid phase in step c) and/or e) byother elution means such as e.g. heating.

According to one embodiment, DNA as well as RNA is bound in step d) to asolid phase and thus is isolated according to the method of the presentinvention. As discussed above, the teachings of the present inventionincrease the overall nucleic acid yield while preserving the integrityof the nucleic acids.

According to one embodiment, the sample comprises at least onenon-target nucleic acid and at least one target nucleic acid and themethod aims at isolating predominantly the target nucleic acid. E.g. thenon-target nucleic acid can be DNA and the target nucleic acid can beRNA or vice versa.

According to one embodiment, isolation step d) comprises severalintermediate steps in order to allow the isolation of predominantly thetarget nucleic acid. According to one embodiment, isolation step d)comprises not only a sample digestion step (see above) but also anintermediate step that removes at least a portion of non-target nucleicacid. Preferably, the non-target nucleic acid is removed by binding atleast a portion of the non-target nucleic acid under appropriateconditions to a solid phase and then separating the non-target nucleicacid bound to the solid phase from the remaining sample comprising thetarget nucleic acid. This can be achieved e.g. by the addition of asuitable solid phase under conditions wherein mainly the non-targetnucleic acids are bound to the solid phase. Suitable methods forselectively removing a non-target nucleic acid from a target nucleicacid are for example described in EP 0 880 537 and WO 95/21849, hereinincorporated by reference. If desired, said non-target nucleic acid mayalso be further used, e.g. further processed such as e.g. eluted fromthe solid phase. However, it may also be discarded. When intending toisolate (only) RNA as target nucleic acid, the non-target nucleic acidis usually DNA.

In order to further reduce the amount of non-target nucleic acids in theisolated target nucleic acid, an intermediate step for degradingnon-target nucleic acids using a suitable enzyme can be performed afterat least the portion of the non-target nucleic acid was removed. It isalso within the scope of the present invention to skip the removal stepand to destroy non-target nucleic acids by using one or more appropriateenzymes only. Thus, according to one embodiment, isolation step d)comprises performing an enzymatic treatment in order to degrade(remaining) non-target nucleic acids. According to one embodimentwherein RNA is isolated as target nucleic acid, a DNase treatment isperformed. As the conditions for performing a DNase digest are wellknown in the prior art, they do not need further description here.Basically the same applies when isolating DNA as target nucleic acid andaccordingly when using an RNase for degrading RNA as non-target nucleicacid.

According to a preferred embodiment of the present invention wherein RNAis isolated from a sample comprising at least RNA and DNA, isolationstep d) comprises the following steps

-   -   i) removing at least a portion of the DNA from the resuspended        and preferably digested sample, by binding DNA to a first solid        phase and separating the DNA bound to said first solid phase        from the remaining sample comprising the RNA,    -   ii) binding the RNA to a second solid phase, wherein at least        one chaotropic agent and at least one alcohol in a concentration        ≥30% v/v is used during this binding step ii),    -   optionally performing at least one washing step for washing the        RNA bound to said second solid phase, and    -   optionally eluting the RNA from said second solid phase.

Isolation step d) may also comprise additional steps, e.g. at least oneadditional enzymatic digestion step to digest remaining DNA and/orprotein contaminations.

According to a preferred embodiment of the present invention wherein atleast RNA is isolated from a sample comprising at least RNA and DNA,isolation step d) comprises the following steps:

-   -   Obtaining the resuspended sample comprising a proteolytic enzyme        and continuing the digestion of the resuspended sample        preferably by incubating the resuspended sample for at least 5        min above room temperature preferably above 50° C. Suitable        incubation conditions are described above, it is referred to the        respective disclosure.    -   Removing at least a portion of the DNA from the resuspended and        digested sample, by binding DNA to a first solid phase and        separating the DNA bound to said first solid phase from the        remaining sample comprising the RNA. Thereby, the DNA can be        removed. The removed DNA can be further processed, e.g. analysed        or amplified. Optionally, the DNA is eluted from the first solid        phase if a parallel isolation of RNA and DNA is of interest.    -   Binding the RNA to a second solid phase, wherein at least one        chaotropic agent and at least one alcohol in a concentration        ≥30% v/v is used during this RNA binding step. Suitable binding        conditions and in particular suitable concentration ranges for        the chaotropic agent and the alcohol are described above, it is        referred to the respective disclosure.    -   Optionally performing at least one washing step for washing the        RNA bound to said second solid phase. Details with respect to        the washing step were described above, it is referred to the        respective disclosure.    -   Optionally performing a DNase digest and/or a digest using a        proteolytic enzyme. Performing a DNase digest has the advantage        that remaining traces of DNA can be efficiently removed.        Performing a second protein digestion step is also advantageous        in order to increase the purity of the isolated RNA. Preferably,        the RNA is eluted prior to performing the DNase digest and the        proteolytic enzyme is added after the DNase digest was performed        and the reaction mixture is incubated in the presence of a        chaotropic agent. Suitable digestion conditions are also        described above. Preferably, proteinase K is used as proteolytic        enzyme. Details with respect to said second protein digestion        step and the associated advantages are described in EP 10 007        346.9. After the protein digestion step was performed, the RNA        is re-bound to the second solid phase preferably by adding at        least one chaotropic agent and at least one alcohol. Suitable        binding conditions are described above, it is referred to the        respective disclosure. Preferably, the same binding conditions        are used that were used in the first RNA binding step. After        rebinding, optionally one or more washing steps can be        performed. Optionally the RNA is eluted from said second solid        phase and optionally the eluted RNA is denatured by performing a        heat treatment.

It is also within the scope of the present invention to performadditional intermediate steps than the ones described herein. However,according to certain embodiments, no additional steps other than theones described herein are performed.

The sample from which the nucleic acids are to be isolated can be,respectively were according to one embodiment stabilised by contactingthe sample with a stabilizing composition having one or more of thefollowing characteristics:

-   -   a) it comprises        -   a) a cationic compound of the general formula:

Y⁺R₁R₂R₃R₄X⁻

-   -   -   -   wherein Y represents nitrogen or phosphor, preferably                nitrogen            -   R₁R₂R₃ and R₄ independently, represent a branched or                unbranched C₁—C₂₀-alkyl group, a C₆-C₂₀_aryl group                and/or a C₆-C₂₆ aralkyl group;            -   X⁻ represents an anion of an inorganic or organic, mono-                or polybasic acid; and

        -   b) at least one proton donor.

According to one embodiment, which is particularly preferred when RNA isisolated from a biological sample such as whole blood or blood productssuch as plasma or serum, the nucleic acids contained in the sample arestabilised preferably immediately after the biological sample has beentaken from its natural environment in order to preserve the status quoof the nucleic acid population comprised in the sample, in particularthe transcription pattern. This is particularly beneficial in themedical and diagnostic field.

Thus, according to one embodiment, after the sample was obtained it ispreferably immediately mixed with a nucleic acid storage stabilizationcomposition for stabilizing nucleic acids in said sample prior toisolating the nucleic acids therefrom. E.g. the sample can be collectedin a suitable collection device, e.g. an evacuated blood collectiontube, which comprises the stabilization composition. Thereby, the sampleis immediately stabilized upon collection. According to one embodiment,said stabilization composition comprises

-   -   a) a cationic compound of the general formula:

Y⁺R₁R₂R₃R₄X⁻

-   -   -   wherein Y represents nitrogen or phosphor, preferably            nitrogen        -   R₁R₂R₃ and R₄ independently, represent a branched or            unbranched C₁—C₂₀-alkyl group, a C₆—C₂₀_aryl group and/or a            C₆—C₂₆ aralkyl group;        -   X⁻ represents an anion of an inorganic or organic, mono- or            polybasic acid; and

    -   b) at least one proton donor, wherein the proton donor is        preferably present in the composition in a concentration of        above 50 mM to saturation and wherein the proton donor is        preferably selected from the group consisting of saturated        aliphatic monocarboxylic acids, unsaturated alkenyl-carboxylic        acids, saturated and/or unsaturated aliphatic C₂—C₆-dicarboxylic        acids, aliphatic hydroxyl-di- and tricarboxylic acids, aliphatic        ketocarboxylic acids, amino acids or the inorganic acids or the        salts thereof, on their own or in combination.

Preferably, R₁ denotes a higher alkyl group with 12, 14 or 16 carbonatoms and R₂, R₃ and R₄ each represent a methyl group.

Preferably, the anion X⁻ represents an anion of hydrohalic acids oranions of mono- or dibasic organic acids, most preferred the anion X⁻ isselected from the group consisting of bromide, chloride, phosphate,sulphate, formate, acetate, propionate, oxalate, malonate, succinate orcitrate.

Preferably, the proton donor is selected from the group consisting ofsaturated aliphatic monocarboxylic acids, unsaturated alkenyl-carboxylicacids, saturated and/or unsaturated aliphatic C₂-C₆-dicarboxylic acids,aliphatic ketocarboxylic acids, amino acids or the inorganic acids orthe salts thereof, and combinations thereof. Preferably, the aliphaticmonocarboxylic acid comprises a C₁-C₆-alkyl-carboxylic acid selectedfrom the group consisting of acetic acid, propionic acid, n-butyricacid, n-valeric acid, isovaleric acid, ethyl-methyl-acetic acid(2-methyl-butyric acid), 2,2-dimethylpropionic acid (pivalic acid),n-hexanoic acid, n-octanoic acid, n-decanoic acid or n-dodecanoic acid(lauric acid) or mixtures thereof. Preferably, the aliphaticalkenyl-carboxylic acid is selected from the group consisting of acrylicacid (propenoic acid), methacrylic acid, crotonic acid, isocrotonic acidor vinylacetic acid or mixtures thereof. Preferably, the saturatedaliphatic C₂-C₆-dicarboxylic acid is selected from the group consistingof oxalic acid, malonic acid, succinic acid, glutaric acid or adipicacid or mixtures thereof. Most preferred, the aliphatic dicarboxylicacid is oxalic acid or succinic acid or mixtures thereof. Preferably,the aliphatic hydroxy-di- and —tricarboxylic acids are selected from thegroup consisting of tartronic acid, D-(+), L-(−) or DL-malic acid, (2R,3R)-(+)-tartaric acid, (2S, 3S)-(−)-tartaric acid, meso-tartaric acidand citric acid or mixtures thereof. Most preferred, the unsaturateddicarboxylic acid is maleic and/or fumaric acid or mixtures thereof.Preferably, the unsaturated tricarboxylic acid is aconitic acid.Preferably, the aliphatic ketodicarboxylic acids are mesoxalic acid oroxaloacetic acid, or mixtures thereof. Preferably, the amino acids areselected from the group consisting of aminoacetic acid (glycine),alpha-aminopropionic acid (alanine), alpha-amino-iso-valeric acid(valine), alpha-amino-iso-caproic acid (leucine) andalpha-amino-beta-methylvaleric acid (isoleucine), or mixtures thereof.

Preferably, the stabilising composition is present in an aqueoussolution. Preferably, the cationic compound is comprised in aconcentration in the range from 0.01 weight percent to 15 weightpercent.

Suitable stabilising solutions are also described in detail e.g. in U.S.Pat. No. 7,270,953, herein incorporated by reference. As describedabove, the cationic compound comprised in the stabilisation compositionforms complexes with nucleic acids comprised in the sample. According toone embodiment, the stabilised sample does not comprise a chelatingagent. According to another embodiment, it may comprise a chelatingagent.

According to an alternative embodiment, the stabilization compositioncomprises a detergent which comprises under the used conditions acharged quarternary ammonium cation as polar head group and thus is orbecomes cationic under the used stabilisation conditions. Accordingly,also an originally non-ionic detergent can be used for providing nucleicacid complexes, if the detergent is or becomes cationic duringstabilisation, respectively complex formation. Thus, besides cationicdetergents comprising a permanently charged head group also anoriginally ternary amine can be used as cationic detergent if providedin an acidic environment whereby the ternary amine incorporates a protonand become positively charged.

According to one embodiment, an amino surfactant having the followingformula 2 is used as detergent in the stabilisation composition which isor becomes cationic under the used stabilisation conditions and therebyfunctions as cationic detergent:

R1R2R3N(O)x   (2)

wherein,

R1 and R2 each independently is H, C1—C20 alkyl residue, C6—C26 arylresidue or C6—C26 aralkyl residue, preferably H, C1—C6 alkyl residue,C6—C12 aryl residue or C6—C12 aralkyl residue,

R3 is C1—C20 alkyl group, C6—C26 aryl residue or C6—C26 aralkyl residue,

X is an integer of 0 and 1.

According to one embodiment, x is 1 and R1 and R2 each independently isC1—C6 alkyl, and R3 is C1-C20 alkyl. According to a preferredembodiment, x is 0. According to one embodiment, said amino surfactantis selected from the group consisting of dodecylamine,N-methyldodecylamine, N,N-dimethyldodecylamine, N,N-dimethyldodecylamineN oxide and 4-tetradecylaniline. Preferably, said amino surfactant iscomprised in a stabilisation composition which additionally comprises atleast one proton donor, preferably an acid or acid salt to render aquarternary ammonium cation. According to one embodiment, thestabilization composition comprises at least one acid salt selected fromthe group consisting of maleic acid, tartaric acid, citric acid, oxalicacid, carboxylic acids and mineral acids. The total concentration ofsaid acid salt in the stabilization composition may preferably rangefrom 0.01 M to 1 M.

Complex samples that are stabilised as is described above using acationic detergent are a particular challenge for isolating nucleicacids due to the additives in the stabilising solution and the highprotein content of the sample, in particular if the sample is a bloodsample or a sample derived from blood. The method of the presentinvention overcomes these difficulties and allows the isolation of RNAincluding small RNA (if desired) with good yield and high purity fromrespectively stabilised samples and in particular from complexbiological samples such as whole blood and blood products such as buffycoat, serum and/or plasma or tissue samples, in particular organ tissuesamples e.g. obtained from lung or liver. Therefore, the method of thepresent invention is particularly useful in the medical and inparticular in the diagnostic field.

According to one embodiment, the resuspended sample comprising thenucleic acid, the at least one chaotropic agent, the at least onechelating agent and optionally the protein-degrading compound is put onhold between step c) and step d). Said holding time prior to isolatingthe nucleic acid in step d) may have a duration of more than 0.1 h, morethan 0.25 h, more than 0.5 h, more than 0.75 h, more than 1 h, more than1.5 h, more than 2 h, more than 2.5 h or more than 3 h hours andpreferably lies in a range selected from 0.25 h to 12 h, 0.5 h to 11 h,0.75 h to 10 h, 1 h to 9 h, 1.25 h to 8.5 h, 1.5 h to 8 h, 1.75 h to 7.5h, 1.25 h to 7 hours, 1.5 h to 6.5 h, 1.75 h to 6 h and 2 h to 5.5 h.

As discussed above, the circumstance that samples are put on holdbetween the resuspension in step c) and the actual nucleic isolationstep d) often occurs when processing a plurality of samples using anautomated system. As discussed above, the samples are often manuallyprepared (e.g. by centrifuging the samples to obtain the pelletcomprising the complexes comprising the cationic detergent and thenucleic acids and adding the resuspension chemistry) for nucleic acidisolation. Afterwards, the prepared, resuspended samples are placed intoa robotic system for nucleic acid isolation. Said robotic systems mayalso execute the digestion step described above as they are oftendesigned to perform heating and/or agitation steps. The method accordingto the present invention has particular advantages when processing aplurality of samples, in particular when using an automated system.

Thus, according to one embodiment, a plurality of samples is processed,wherein the holding time between step c) and step d) differs at leastbetween some of the resuspended samples. According to this embodiment, aplurality of samples is processed according to the present method up tostep c). After resuspension in step c), a portion of said plurality ofsamples is further processed according to step d), while the remaining,resuspended samples are put on hold. These resuspended samples that wereput on hold are then further processed according to step d), preferablyafter the nucleic acids were isolated from the first portion ofresuspended samples.

Preferably, the plurality of samples is processed using an automatedsystem which is capable of processing magnetic particles. Using magneticparticles to isolate nucleic acids from the resuspended sample in stepd) is beneficial because of the simplified handling. Magnetic particlescan be processed by the aid of a magnetic field. Suitable and preferredmagnetic particles are described above. Here, different automatedrobotic systems exist in the prior art that can be used in conjunctionwith the present invention to process magnetic particles during nucleicacid isolation in step d). According to one embodiment, magneticparticles are collected at the bottom or the side of the reaction vesseland the remaining liquid sample is removed from the reaction vessel,leaving behind the collected magnetic particles to which the nucleicacids are bound. Removal of the remaining sample can occur bydecantation or aspiration. Such systems are well known in the prior artand thus need no detailed description here. In an alternative systemthat is known for processing magnetic particles the magnet which isusually covered by a cover or envelope plunges into the reaction vesselto collect the magnetic particles. The magnetic particles that carry thebound nucleic acids can then be transferred for example into a newreaction vessel e.g. comprising further processing solutions such ase.g. a washing solution. As respective systems are well-known in theprior art and are also commercially available (e.g. QIAsymphony;QIAGEN), they do not need any detailed description here. In a furtheralternative automated system that is known for processing magneticparticles, the sample comprising the magnetic particles can be aspiratedinto a pipette tip and the magnetic particles can be collected in thepipette tip by applying a magnet e.g. to the side of the pipette tip.The remaining sample can then be released from the pipette tip while thecollected magnet particles which carry the bound nucleic acids remaindue to the magnet in the pipette tip. The collected magnetic particlescan then be processed further. Such systems are also well-known in theprior art and are also commercially available (e.g. BioRobot EZ1,QIAGEN) and thus, do not need any detailed description here.

The term “sample” is used herein in a broad sense and is intended toinclude a variety of sources that contain nucleic acids. The sample maybe a biological sample but the term also includes other, e.g. artificialsamples which comprise nucleic acids. Exemplary samples include, but arenot limited to, body fluids in general, whole blood; serum; plasma; redblood cells; white blood cells; buffy coat; swabs, including but notlimited to buccal swabs, throat swabs, vaginal swabs, urethral swabs,cervical swabs, throat swabs, rectal swabs, lesion swabs, abcess swabs,nasopharyngeal swabs, and the like; urine; sputum; saliva; semen;lymphatic fluid; liquor; amniotic fluid; cerebrospinal fluid; peritonealeffusions; pleural effusions; fluid from cysts; synovial fluid; vitreoushumor; aqueous humor; bursa fluid; eye washes; eye aspirates; plasma;serum; pulmonary lavage; lung aspirates; and tissues, including but notlimited to, liver, spleen, kidney, lung, intestine, brain, heart,muscle, pancreas, cell cultures, as well as lysates, extracts, ormaterials obtained from any cells and microorganisms and viruses thatmay be present on or in a sample and the like. Materials obtained fromclinical or forensic settings that contain nucleic acids are also withinthe intended meaning of the term sample. Furthermore, the skilledartisan will appreciate that lysates, extracts, or materials or portionsthereof obtained from any of the above exemplary samples are also withinthe scope of the term sample. Preferably, the sample is a biologicalsample derived from a human, animal, plant, bacteria or fungi. Inparticular, the term “sample” refers to a nucleic acid containing samplewhich also comprises proteins. Preferably, the sample is selected fromthe group consisting of cells, tissue, bacteria, virus and body fluidssuch as for example blood, blood products such as buffy coat, plasma andserum, urine, liquor, sputum, stool, CSF and sperm, epithelial swabs,biopsies, bone marrow samples and tissue samples, preferably organtissue samples such as lung and liver. Preferably, the sample isselected from whole blood and blood products such as buffy coat, serumor plasma.

The term “nucleic acid” or “nucleic acids” as used herein, in particularrefers to a polymer comprising ribonucleosides and/ordeoxyribonucleosides that are covalently bonded, typically byphosphodiester linkages between subunits, but in some cases byphosphorothioates, methylphosphonates, and the like. Nucleic acidsinclude, but are not limited to all types of DNA and/or RNA, e.g. gDNA;circular DNA; circulating DNA; hnRNA; mRNA; noncoding RNA (ncRNA),including but not limited to rRNA, tRNA, IncRNA (long non coding RNA),lincRNA (long intergenic non coding RNA), miRNA (micro RNA), siRNA(small interfering RNA), snoRNA (small nucleolar RNA), snRNA (smallnuclear RNA) and stRNA (small temporal RNA), piRNA (piwi interactingRNA), tiRNA (transcription initiation RNA), PASR (promoter associatedRNA), CUT (cryptic unstable transcripts), extracellular or circulatingRNA; fragmented nucleic acid; nucleic acid obtained from subcellularorganelles such as mitochondria or chloroplasts; and nucleic acidobtained from microorganisms, parasites, or DNA or RNA viruses that maybe present in a biological sample. Synthetic nucleic acid sequences thatmay or may not include nucleotide analogs that are added or “spiked”into a biological sample are also within the scope of the invention.Small RNA or the term small RNA species in particular refers to RNAhaving a length of less than 500 nt, 400 nt, 300 nt or 100 nt andincludes but is not limited to miRNA, siRNA, other short interferingnucleic acids, snoRNAs and the like. According to one embodiment, thenucleic acid is RNA. The RNA may include small RNA species.

As becomes apparent from the described examples of samples that can beprocessed according to the method of the present invention, a sample maycomprise more than one type of nucleic acid. Depending on the intendeduse, it may be desirous to isolate all types of nucleic acids from asample ((e.g. DNA and RNA) or only certain types or a certain type ofnucleic acid (e.g. only RNA but not DNA or vice versa or DNA and RNA aresupposed to be obtained separately). All these variants are within thescope of the present invention. Suitable methods for isolating eitherDNA or RNA or both types of nucleic acids in parallel are known in theprior art and are also described above.

The present invention also pertains to a method for isolating nucleicacids from a sample, preferably a blood sample, wherein the nucleicacids are isolated from a plurality of samples and wherein variations inyield and quality of the nucleic acids that are isolated from saidplurality of samples which result from that the plurality of samplesprepared for isolation have diverging holding times before the nucleicacids are isolated from the prepared samples are thereby reduced thatthe samples prepared for isolation comprise at least one chaotropicagent and at least one chelating agent.

The sample is preferably stabilised by using a cationic detergent whichforms a complex with the nucleic acid contained in the sample. Detailswith respect to the cationic detergent are described above, it isreferred to the above disclosure. Preferably, the nucleic acid is RNAand the sample is or is derived from a body fluid, preferably wholeblood, serum or plasma, most preferred whole blood. According to oneembodiment, the nucleic acids are isolated according to the methoddescribed above. It is referred to the above disclosure which alsoapplies here.

Furthermore, the present invention pertains to the use of a chelatingagent for preventing the formation of a precipitate that interacts withthe container wall of a sample comprising a chaotropic agent and anucleic acid. The sample is preferably stabilised by using a cationicdetergent which forms a complex with the nucleic acid contained in thesample. Details with respect to the cationic detergent are describedabove, it is referred to the above disclosure. Preferably, the nucleicacid is RNA and the sample is or is derived from a body fluid,preferably whole blood, serum or plasma, most preferred whole blood. Theadvantages of a respective use of a chaotropic agent and a chelatingagent in particular with respect to the increase in nucleic acid qualityand quantity due to the prevention of the precipitate formation and/oradherence to the container wall were described in detail above. Suitableand preferred embodiments of the chelating agent and concentrationsthereof, the chaotropic agent and concentrations thereof, the cationicdetergent, the nucleic acids and sample types are described above. It isreferred to the above disclosure which also applies here.

This invention is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this invention. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects or embodiments of this inventionwhich can be read by reference to the specification as a whole. The term“solution” as used herein, in particular refers to a liquid composition,preferably an aqueous composition. It may be a homogenous mixture ofonly one phase but it is also within the scope of the present inventionthat a solution that is used according to the present inventioncomprises solid components such as e.g. precipitates. According to oneembodiment, subject matter described herein as comprising certain stepsin the case of methods or as comprising certain ingredients in the caseof compositions, solutions and/or buffers refers to subject matterconsisting of the respective steps or ingredients. It is preferred toselect and combine preferred embodiments described herein and thespecific subject-matter arising from a respective combination ofpreferred embodiments also belongs to the present disclosure.

EXAMPLES

The present invention is now illustrated by the following non-limitingexamples:

Example 1

24 blood samples (2.5 ml) were collected per batch in PAXgene Blood RNATubes (which comprise a cationic detergent as stabilising agent). Thesamples were stabilized for 24 h at room temperature and were processedwith the QIAsymphony PAXgene Blood RNA Kit, 2008 (QIAGEN) according tothe manufacturer's instructions.

QIAsymphony PAXgene Blood RNA Kit

In the QIAsymphony PAXgene Blood RNA protocol (prior art), thestabilised samples are manually prepared in order to collect andresuspend the pellet which comprises the complexes comprising thecationic detergent and the nucleic acids. Said pellet is obtained bycentrifugation and the supernatant is decanted. Afterwards, 300 μl ofthe resuspension buffer BR1 (QIAGEN) was added to the pellet andvortexed. The respectively manually prepared samples were then loaded onthe QIAsymphony robotic system and the nucleic acids were isolated fromthe resuspended samples according to the QIAsymphony script.

The first batch was directly processed after resuspension, the secondbatch after 2 hours 5 minutes. Thus, the samples of the second batch hada 2 hours 5 minutes longer holding time.

Modified Version Comprising the Addition of a Chaotropic Agent DuringResuspension

The QIAsymphony PAXgene Blood RNA protocol was modified by adding achaotropic agent to the resuspended sample in order to preserve the RNAintegrity. The blood samples were manually prepared as described above.After the resuspension buffer BR1 was added to the pellet and vortexed,200 μl of a further solution (BR2, QIAGEN) was added which comprises achaotropic salt (GITC). The chaotropic salt was added to preserve theRNA integrity. The samples were then processed according to twodifferent versions. According to version 1, the samples were directlyloaded on the QIAsymphony robot after the buffer BR2 was added.According to version 2, the samples were vortexed after the addition ofthe buffer BR2 and then loaded on the QIAsymphony robot. In theQIAsymphony robotic system, the nucleic acids were then isolated fromthe resuspended samples according to the QIAsymphony script.

The quality of the isolated RNA was determined with the AgilentBioanalyzer and expressed in RIN values (RIN=RNA integrity number).

Results

The results are shown in FIG. 1:

1: QIAsymphony PAXgene Blood RNA—first batch

2: QIAsymphony PAXgene Blood RNA—second batch

3: QIAsymphony PAXgene Blood RNA modified (addition of chaotropicagent)—second batch (no vortexing)

4: QIAsymphony PAXgene Blood RNA modified (addition of chaotropicagent)—second batch (vortexing)

FIG. 1 shows that the RNA integrity is excellent with the first batch ofthe samples that were processed according to the prior art protocol.However, the RNA integrity decreases in the second batch which had alonger holding time between resuspension of the sample and nucleic acidextraction. Thus, longer holding times decrease the RNA integrity andhence the quality of the RNA.

The modified versions, wherein a chaotropic salt is added to theresuspended samples show no differences with respect to the RNA qualitybetween batch 1 and batch 2. Thus, the RNA integrity is preserved evenif the samples have a long holding time of two hours and more.

Example 2

In Example 2, the QIAsymphony PAXgene Blood RNA protocol described aboveand a modified version (wherein a chaotropic agent is added duringresuspension) was analysed with respect to the nucleic acid yield. Insaid modified version 3 that was tested in Example 2, the nucleic acidisolation was also performed on the QIAsymphony according to theQIAsymphony PAXgene Blood RNA protocol, however, using a modifiedbinding buffer in order to improve nucleic acid binding, in particularthe binding of small RNA. The modified binding buffer that was used inExample 2 instead of the binding buffer QSB1 that is used in theQIAsymphony PAXgene Blood RNA protocol, comprised more than 60%isopropanol and 0.2 M sodium trichloroacetate. Respectively modifiedbinding buffers to achieve improved binding in particular of smallnucleic acids and corresponding nucleic acid isolation protocols aredescribed in EP 10 000 432.4, herein incorporated by reference. Theresuspension of the sample was performed as described in Example 1(modified version) and hence, a chaotropic agent (GITC) was added inform of buffer BR2 to preserve the RNA integrity.

For the respectively modified version 3, 3 batches were tested. Thefirst batch of samples was directly processed after sample resuspension,the second batch had a holding time of 2 hours 5 minutes, the thirdbatch had a holding time of 4 hours 10 minutes. As additional reference,samples were also processed with the PAXgene Blood RNA Kit (a membranebased, single spin column method) according to the manufacturersinstructions. The results of Example 2 are shown in FIG. 2:

1: QIAsymphony PAXgene Blood RNA

2: QIAsymphony PAXgene Blood RNA modified (addition of chaotropicagent)—first batch

3: QIAsymphony PAXgene Blood RNA modified (addition of chaotropicagent)—second batch

4: QIAsymphony PAXgene Blood RNA modified (addition of chaotropicagent)—third batch

5: PAXgene Blood RNA (IVD)

As can be derived from FIG. 2, the first batch of the modified versionachieves good yields which are also improved compared to the prior artmethod. However, reduced yields are observed for the 2 ^(nd) and 3 ^(rd)batch with the modified version, wherein the chaotropic agent is addedto the resuspended sample to guarantee high RIN values (see Example 1and FIG. 1). The yield is reduced up to 20 to 30%. The reason for thisloss in nucleic acid yield is that a precipitate is formed during theholding time which sticks tightly to the plastic wall of the tube.

Example 3

In order to ensure both, a high RNA integrity and a high nucleic acidyield, the protocol was modified according to the teachings of thepresent invention. Thus, the pellet was resuspended by adding a modifiedresuspension buffer, which additionally comprised EDTA. For thispurpose, 25 mM EDTA was added to the resuspension buffer BR1 (QIAGEN)that is used for resuspension in the prior art protocol. 280 μl of thismodified resuspension buffer BR1 was added to the pellet that wasobtained from the PAXgene stabilised blood samples (see Example 1).Furthermore, for resuspension 20 μl proteinase K was added per sample.The sample was resuspended by vortexing and 200 μl of a buffercomprising a chaotropic agent (BR2, QIAGEN—comprises>3 M GITC) wasadded.

The resuspended samples were loaded on the QIAsymphony robotic systemand the automated nucleic acid extraction was performed. In brief, thenucleic acid isolation protocol comprised the following steps:

1. Sample digestion

40 μl proteinase K was added to the resuspended sample and incubated for10 min under heating and shaking.

2. DNA removal

Mag Attract G beads (QIAGEN) were added to the digested sample to bindthe DNA to the beads. The beads were than removed from the sample.

3. RNA binding

Mag Attract G beads (QIAGEN) were added and 1500 μl of a binding buffercomprising more than 65% isopropanol, sodium trichloroacetate and a Trisbuffer.

4. Washing

Two washing steps were performed using the washing buffers QSB1 and BR4(QIAGEN).

5. DNase and Proteinase K digest

The RNA was eluted and DNA traces were digested using DNase I.Afterwards, a second proteinase K digest was performed (see EP 10 007346.9, herein incorporated by reference).

6. RNA Re-binding

1400 μl of the binding buffer (see above) was added.

7. Washing

4 final washing steps were performed (using the buffers QSB1, QSW5 andBR4, all QIAGEN).

8. Elution and heat denaturation

The RNA was eluted using 200 μl BR5 (QIAGEN) and the eluted nucleic acidwas heat denatured for 10 min.

24 blood samples were collected per batch in PAXgene Blood RNA Tubes.This protocol according to the present invention was compared to themodified version as described in example 2 (wherein a chaotropic agentwas added during resuspension to preserve the RNA integrity but noEDTA). Three batches were tested in each method. The first batch wasdirectly processed after sample resuspension, the second batch had aholding time of 2 hours 20 minutes hours, the third batch had a holdingtime of 4 hours 40 minutes hours. As additional reference, samples wereprocessed with the PAXgene Blood RNA kit (membrane based, single spincolumn method).

The results are shown in FIGS. 3 to 6.

FIG. 3 shows the effect of the modified resuspension step according tothe present invention on the precipitate formation. On the left handside, the tubes of the 3 ^(rd) batch are shown wherein the samples wereprepared according to the modified version (see example 2), wherein achaotropic agent is added during resuspension (but no EDTA). As can beseen, a large precipitate is formed which irreversibly sticks to thecontainer walls and thus, is lost for the subsequent nucleic acidisolation (=reduced yield, see example 2). On the right hand side, thetubes of the 3 ^(rd) batch are shown wherein the samples were preparedaccording to the method of the present invention, wherein EDTA is addedduring resuspension in addition to the chaotropic agent. As can be seen,the precipitate formation is substantially prevented with the methodaccording to the present invention.

FIG. 4 shows the overall RNA yield that is obtained with differentbatches of the protocols under comparison:

1: QIAsymphony PAXgene Blood RNA—first batch

2: QIAsymphony PAXgene Blood RNA modified according to theinvention—first batch

3: QIAsymphony PAXgene Blood RNA—second batch

4: QIAsymphony PAXgene Blood RNA modified according to theinvention—second batch

5: QIAsymphony PAXgene Blood RNA—third batch

6: QIAsymphony PAXgene Blood RNA modified according to theinvention—third batch

7: PAXgene Blood RNA (IVD)

FIG. 4 demonstrates that the samples that are processed according to themethod of the present invention achieve constant high nucleic acidyields despite longer holding times between the different batches. Thenucleic acid yield is considerably improved.

FIG. 5 shows the RNA integrity that is achieved with the methodaccording to the present invention. As can be seen, a high RIN isobtained with all three batches. Thus, the integrity of the RNA ispreserved, despite long holding times.

FIG. 6 demonstrates the achieved yield of small RNAs:

1: QIAsymphony PAXgene Blood RNA—first batch

2: QIAsymphony PAXgene Blood RNA modified according to theinvention—first batch

3: QIAsymphony PAXgene Blood RNA—second batch

4: QIAsymphony PAXgene Blood RNA modified according to theinvention—second batch

5: QIAsymphony PAXgene Blood RNA—third batch

6: QIAsymphony PAXgene Blood RNA modified according to theinvention—third batch

7: PAXgene Blood RNA (IVD)

FIG. 6 shows the mean C_(T) obtained with the miScript® Primer assay(hsa-miR 30b) when analysing the eluates obtained with the differenttested protocols. As can be seen, the method achieves a good yield ofsmall RNAs which is also considerably improved compared to the prior artmethod.

Example 4

In example 4, the effects of the addition of EDTA during resuspension onthe RNA yield was further analysed. Optionally, a protein-degradingcompound (proteinase K) was added. For each test series, 24 test tubes(PAXgene Blood RNA Tubes from QIAGEN) filled with 2.5 ml whole humanblood were obtained in duplicates from 12 individual donors and thenucleic acid containing pellet was obtained. The supernatants wereremoved, and the pellets were resuspended by vortexing in thefresuspension buffer BR1 (QIAGEN):

280 μl BR1 (QIAGEN), 20 μl proteinase K solution (QIAGEN) and 200 μl ofa GITC containing buffer (BR2, QIAGEN);

280 μl BR1 (QIAGEN)+10 mM EDTA, 20 μl proteinase K solution (QIAGEN) and200 μl of a GITC containing buffer (BR2, QIAGEN);

280 μl BR1 (QIAGEN)+25 mM EDTA, 20 μl proteinase K solution (QIAGEN) and200 μl of a GITC containing buffer (BR2, QIAGEN);

280 μpl BR1 (QIAGEN)+50 mM EDTA, 20 μl proteinase K solution (QIAGEN)and 200 μl of a GITC containing buffer (BR2, QIAGEN);

280 μl BR1 (QIAGEN)+25 mM EDTA, 20 μl proteinase K solution (QIAGEN) and200 μl of a GITC containing buffer (BR2, QIAGEN); or 300 μl BR1(QIAGEN)+25 mM (no proteinase K) and 200 μl of a GITC containing buffer(BR2, QIAGEN).

The samples were processed according to the protocol described inExample 3.

FIG. 7 shows the mean total RNA yield. For samples resuspended in bufferBR1 with proteinase K and the chaotropic agent but no EDTA, the RNAyield achieved from batch 1 samples (with short holding time) was higherthan for batch 2 samples (with extended holding time). Samples that wereresuspended using a chaotropic agent, proteinase K and the chelatingagent EDTA generally yielded more RNA as can be seen from the nucleicacid yields that are obtained with the first batch. Thus, the RNA yieldwas generally improved. Furthermore, the RNA yield that is obtained fromthe second batch of samples after an extended holding time is comparableor—at higher EDTA concentrations (25 mM and 50 mM)—even better than theRNA yield that is obtained with the first batch. Similar results areachieved if the proteinase K is omitted during resuspension. Thus, thebeneficial effect on the RNA yield is attributable to the addition ofthe chelating agent.

Example 5

As explained above, the resuspended samples form precipitates duringlonger holding times, in particular if a chaotropic agent is addedduring resuspension. Precipitated aggregates remain stuck to the testtube after RNA-processing and are therefore not included in thepurification procedure. As a consequence, blood samples of later batchesshow more pronounced precipitation than samples from earlier batches.Example 5 shows that the precipitate formation and adherence to thecontainer wall that reduces the RNA yield (see above) occursirrespective of the container material and thus in glass tubes as wellas in plastic tubes.

Samples of stabilized whole human blood (using the PAXgene stabilisationchemistry) were processed in glass or plastic tubes. The stabilizedsamples were centrifuged, the supernatants were removed, and the pelletswere resuspended by vortexing in a mixture of 300 μl buffer BR1 and 200μl buffer BR2. Furthermore, the resuspension protocol according to thepresent invention was tested, wherein additionally 25 mM EDTA was addedduring resuspension.

The resuspended samples were left aside for 4 hours. Then, thesupernatants were removed and the precipitates were documented in bothglass- and plastic-test tubes.

The results are shown in FIG. 8A and FIG. 8B. For both materials tested,bulk precipitates were found stuck at the test tube bottom for thesamples that were resuspended without EDTA (see FIG. 8A), representinglost material that will not be included in the subsequent RNA isolationprocedure and thus results in reduced nucleic acid yields. Samples thatwere resuspended according to the method according to the presentinvention, wherein accordingly, EDTA was additionally included duringresuspension, no precipitates were found even after a holding time of 4hours.

1. A method for isolating nucleic acids from a sample, preferably ablood sample, comprising the following steps: a) obtaining a samplewhich has been stabilised by the use of at least one cationic detergent,wherein the cationic detergent has formed complexes with the nucleicacids; b) obtaining the complexes optionally together with other samplecomponents from the stabilised sample, wherein said complexes comprisethe nucleic acids to be isolated; c) resuspending the complexes andoptionally adding one or more additives before, during and/or afterresuspension, thereby obtaining a resuspended sample comprising at leasti) the nucleic acid to be isolated; ii) at least one chaotropic agent;and iii) at least one chelating agent; and d) isolating nucleic acidsfrom the resuspended sample.
 2. The method according to claim 1, whereinthe resuspended sample is put on hold between step c) and step d) for atleast 0.2 h, at least 0.3 h, at least 0.4 h, at least 0.5 h, at least0.75 h or at least 1 h and/or for a time period of 0.5 h to 12 h, 1 h to10 h, 1.5 h to 8 h, 2 h to 7 h or 3 h to 6 h.
 3. The method according toclaim 1, wherein the resuspension performed in step c) has one or moreof the following characteristics: a) a resuspension solution is addedwherein said resuspension solution comprises a non-chaotropic salt,preferably an ammonium salt; b) the chelating agent is added prior,during or after resuspension; c) the chelating agent is added separatelyfrom the resuspension solution; d) the chelating agent is comprised in aresuspension solution; e) the chelating agent is added in aconcentration so that the resuspended sample comprises the chelatingagent in a concentration selected from 0.5 mM to 75 mM, 1 mM to 50 mM,2.5 mM to 25 mM and 5 to 15 mM; f) the chelating agent is selected fromthe group consisting of diethylenetriaminepentaacetic acid (DTPA),ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraaceticacid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA) and/or g) at leastone additive is added before, during and/or after resuspension which isselected from the group consisting of chaotropic agents,protein-degrading compounds and buffering agents and thereby iscomprised in the resuspended sample.
 4. The method according to claim 1,wherein the chelating agent present in the resuspended sample a) reducesbinding of the precipitated sample to the container comprising thesample; b) increases the yield of the isolated nucleic acid; and/or c)reduces variations in the nucleic acid isolation efficiency or quantityattributable to different holding times between step c) and d).
 5. Themethod according to claim 1, wherein said method has with respect to thechaotropic agent comprised in the resuspended sample one or more of thefollowing characteristics: a) the concentration of the chaotropic agentin the resuspended sample is selected from the group consisting of 0.1 Mto 4 M, 0.5 M to 3 M and 0.75 M to 2.5 M and preferably is at least 1 M;b) the chaotropic agent is added in step c) in form of a separatesolution; c) the chaotropic agent is added in step c) after resuspensionof the complexes and thereby is comprised in the resuspended sample;and/or d) the chaotropic agent present in step c) is selected from thegroup consisting of chaotropic salts, guanidinium hydrochloride,guanidinium thiocyanate, guanidinium isothiocyanate, sodium thiocyanate,sodium iodide, sodium perchlorate, sodium trichloroacetate, sodiumtrifluroacetate, urea and preferably is GTC or GITC.
 6. The methodaccording to claim 1, wherein a protein degrading compound is added instep c).
 7. The method according to claim 6, wherein said method haswith respect to the protein degrading compound that is added in step c)one or more of the following characteristics: a) the protein degradingcompound is a proteolytic enzyme; and/or b) the protein degradingcompound is a proteolytic enzyme selected from the group consisting ofproteinases, proteases, subtilisins and subtilases, and preferably isproteinase K.
 8. The method according to claim 1, wherein step c)comprises aa) a resuspension solution is added which comprises anon-chaotropic salt and a chelating agent and which does not comprise achaotropic agent and the complexes are resuspended, bb) a chaotropicagent is added after resuspension of the complexes and thereby becomescomprised in the resuspended sample wherein preferably, the chaotropicagent is added to the resuspended complexes in form of an aqueoussolution, cc) optionally a proteolytic enzyme is added afterresuspension of the complexes and thereby is included in the resuspendedsample.
 9. The method according to claim 1, wherein the isolationperformed in step d) comprises the following steps: i) digesting and/ordenaturing the resuspended sample, preferably by heating and/oragitating the resuspended sample in the presence of a proteolyticenzyme; ii) binding the nucleic acids to a solid phase using appropriatebinding conditions, and iii) optionally washing the nucleic acids; iv)optionally eluting the nucleic acids.
 10. The method according to claim1, wherein the sample is a blood sample and in step a) the blood sampleis stabilised by contacting the blood sample with a stabilizingcomposition comprising i) a cationic compound of the general formula:Y⁺R₁R₂R₃R₄X⁻ wherein Y represents nitrogen or phosphor, preferablynitrogen R₁R₂R₃ and R₄ independently, represent a branched or unbranchedC₁-C₂₀-alkyl group, a C₆-C₂₀-aryl group and/or a C₆-C₂₆ aralkyl group;X⁻ represents an anion of an inorganic or organic, mono- or polybasicacid; and ii) at least one proton donor.
 11. The method according toclaim 1, wherein the sample is a blood sample and in step a) the bloodsample is stabilised by contacting the blood sample with a stabilizingcomposition comprising (i) an amino surfactant having the followingformula (2):R1R2R3N(O)x (2) wherein, R1 and R2 each independently is H, C1-C6 alkylresidue, C6-C12 aryl residue or C6-C12 aralkyl residue, R3 is C1-C20alkyl group, C6-C26 aryl residue or C6-C26 aralkyl residue, X is aninteger of 0 and 1 and (ii) an acid or acid salt.
 12. A method accordingto claim 1, wherein a plurality of samples is processed and wherein theholding time between step c) and step d) differs at least between someof the resuspended samples.
 13. The method according to claim 1, havingone or more of the following characteristics: a) the nucleic acid isRNA; b) a plurality of samples is prepared according to steps a) to c)thereby providing a plurality of resuspended samples, wherein theresuspended samples are divided into batches and the nucleic acids areisolated from the batches according to step d) and wherein the holdingtime between step c) and d) varies at least between two batches; c) stepd) is performed using an automated system; d) wherein a plurality ofsamples is processed manually up to step c) thereby providing aplurality of resuspended samples and wherein preferably the resuspendedsamples are processed using an automated system for isolating thenucleic acids in step d); and/or e) at least RNA is isolated from asample comprising at least RNA and DNA and wherein isolation step d)comprises the following steps obtaining the resuspended samplecomprising a proteolytic enzyme and continuing the digestion of theresuspended sample preferably by incubating the resuspended sample forat least 5 min above room temperature preferably above 50° C.; removingat least a portion of the DNA from the resuspended and digested sample,by binding DNA to a first solid phase and separating the DNA bound tosaid first solid phase from the remaining sample comprising the RNA,binding the RNA to a second solid phase, wherein at least one chaotropicagent and at least one alcohol in a concentration>30% v/v is used duringthis RNA binding step, optionally performing at least one washing stepfor washing the RNA bound to said second solid phase, and optionallyperforming a DNase digest and/or a digest using a proteolytic enzyme,optionally eluting the RNA.
 14. A method according to claim 1, forisolating RNA from a blood sample, comprising the following steps: a)obtaining a sample which has been stabilised by the use of at least onecationic detergent, wherein the cationic detergent has formed complexeswith the nucleic acids and wherein preferably, a cationic detergent asdefined in claim 10 or 11 was used for stabilisation; b) obtaining thecomplexes optionally together with other sample components from thestabilised sample, wherein said complexes comprise the nucleic acids tobe isolated; c) resuspending the complexes and optionally adding one ormore additives before, during and/or after resuspension, wherein step c)comprises: aa) adding a resuspension solution which comprises anon-chaotropic salt and a chelating agent wherein the resuspensionsolution does not comprise a chaotropic salt and resuspending thecomplexes, bb) adding a chaotropic agent after resuspension of thecomplexes wherein preferably, the chaotropic agent is added to theresuspended complexes in form of an aqueous solution, cc) optionallyadding a proteolytic enzyme after resuspension of the complexes, therebyobtaining a resuspended sample comprising at least i) the nucleic acidto be isolated; ii) at least one chaotropic agent; and iii) at least onechelating agent; and iv) optionally a proteolytic enzyme; and d)isolating RNA from the resuspended sample, wherein preferably, the RNAis isolated according to the features of claims 13e) and whereinpreferably, magnetic silica particles are used as solid phase forbinding the RNA and wherein preferably, an automated system is used forRNA isolation, and wherein a plurality of samples is prepared accordingto steps a) to c) thereby providing a plurality of resuspended samples,wherein the resuspended samples are divided into batches and the nucleicacids are isolated from the batches according to step d) and wherein theholding time between step c) and d) varies at least between two batches.15. A method for isolating nucleic acids from a sample, preferably ablood sample, wherein the nucleic acids are isolated from a plurality ofsamples and wherein variations in yield and quality of the nucleic acidsthat are isolated from said plurality of samples which result from thatthe plurality of samples prepared for isolation have diverging holdingtimes before the nucleic acids are isolated from the prepared samplesare thereby reduced that the samples prepared for isolation comprise atleast one chaotropic agent and at least one chelating agent.
 16. Themethod according to claim 15, wherein the nucleic acids are isolatedaccording to a method that comprises the following steps: a) obtaining asample which has been stabilised by the use of at least one cationicdetergent, wherein the cationic detergent has formed complexes with thenucleic acids; b) obtaining the complexes optionally together with othersample components from the stabilised sample, wherein said complexescomprise the nucleic acids to be isolated; c) resuspending the complexesand optionally adding one or more additives before during and/or afterresuspension, thereby obtaining a resuspended sample comprising at leasti) the nucleic acid to be isolated; ii) at least one chaotropic agent;and iii) at least one chelating agent; and d) isolating nucleic addsfrom the resuspended sample.
 17. Use of a chelating agent in order toprevent or reduce the formation of a precipitate that attaches to thecontainer wall of a sample comprising at least one chaotropic agent andnucleic acids.
 18. The use according to claim 17, wherein the samplecomprises at least one ingredient selected from the group of a cationicdetergent, a protein-degrading compound, a salt and/or a buffer.